Under normal circumstances, platelet aggregation at sites of vascular trauma results in primary hemostasis. Platelet aggregates may also form on abnormal blood vessels and on prosthetic devices in the circulation resulting in pathologic thrombi. Platelet aggregates form when fibrinogen binds to receptors on the surface of adjacent platelets. There is considerable evidence from a number of laboratories that the platelet membrane glycoprotein llb-llla complex contains the fibrinogen receptor. However, the fibrinogen binding site on llb- llla is cryptic and is only exposed when platelets are stimulated. The goal of this project is to gain an understanding of platelet aggregation by studying the factors involved in the interaction of fibrinogen with the platelet fibrinogen receptor. To study the structure of the platelet glycoproteins llb and llla, we are determining their primary sequences by isolating full-length cDNAs. Because we found it difficult to use human megakaryocytes for this purpose, we have used mRNA from the HEL cell to construct the cDNA. The HEL cell line constitutively expresses proteins nearly identical to platelet llb and lla. The cDNA have been inserted into the bacteriophage expression vector lambda gt11 and this bacteriophage library has been screened to identify clones containing llb cDNA. Additional screens to identify llla cDNA will be performed. Analysis of the necleotide sequences of these cDNA will reveal the llb and llla amino acid sequences. In turn, the amino acid sequences can be used to make predictions about functional domains in the llb-llla complex. To determine which portions of fibrinogen bind to llb- llla, we will study the interaction of peptides corresponding to the twelve C-terminal amino acids of the fibrinogen gamma chain and peptides corresponding to a region near the C-terminus of the fibrinogen alpha chain with activated platelets. The concerted ability of these peptides to inhibit fibrinogen binding to platelets will be tested. Photoaffinity labeling will also be performed to identify the peptide binding sites on llb-llla. Finally, to complement and verify our predictions about the conformation the fibrinogen receptor, we will examine the llb-llla complex with the electronmicroscope. Because llb and llla denature easily, we will attempt to maintain the native conformation of the complex by interacting it with fibrinogen absorbed to a surface. Electronmicroscopic images of adherent llb-llla:fibrinogen complexes then will be obtained.